Proliferation Of Human Pulmonary Artery Smooth Muscle Cells Research Articles

CircST6GAL1 knockdown alleviates pulmonary arterial hypertension by regulating miR-509-5p/multiple C2 and transmembrane domain containing 2 axis.

Hypertension is a main contributing factor of cardiovascular diseases; deregulated circular RNAs are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Herein, we evaluated the function and mechanism of circST6GAL1 in PAH process. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in hypoxic environment for functional analysis. The cell counting kit-8, 5-ethynyl-2'-deoxyuridine, wound healing, and flow cytometry assays were used to investigate cell proliferation, migration, and apoptosis. qRT-PCR and Western blotting analyses were used for level measurement of genes and proteins. The binding between miR-509-5p and circST6GAL1 or multiple C2 and transmembrane domain containing 2 (MCTP2) was analyzed by dual-luciferase reporter, RNA immunoprecipitation, and pull-down assays. The monocrotaline (MCT)-induced PAH mouse models were established for in vivo assay. CircST6GAL1 was highly expressed in PAH patients and hypoxia-induced HPASMCs. Functionally, circST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs. Mechanistically, circST6GAL1 directly targeted miR-509-5p, and MCTP2 was a target of miR-509-5p. Rescue assays showed that the regulatory effects of circST6GAL1 deficiency on hypoxia-induced HPASMCs were abolished. Moreover, forced expression of miR-509-5p suppressed HPASMC proliferation and migration and induced cell apoptosis under hypoxia stimulation, while these effects were abolished by MCTP2 overexpression. Moreover, circST6GAL1 silencing improved MCT-induced pulmonary vascular remodeling and PAH. CircST6GAL1 deficiency reversed hypoxia-induced proliferation and migration, as well as apoptosis arrest in HPASMCs, and alleviated pulmonary vascular remodeling in MCT-induced PAH mouse models through the miR-509-5p/MCTP2 axis, indicating a potential therapeutic target for PAH.

FAK mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation by modulating mitochondrial transcription termination factor 1/cyclin D1.

This study aimed to investigate the mechanism of FAK-dependent hypoxia-induced proliferation on human pulmonary artery smooth muscle cells (HPASMCs). Primary HPASMCs were isolated and cultured invitro under normal and hypoxia conditions to assess cell proliferation with cell counting kit-8. FAK and mitochondrial transcription termination factor 1 (mTERF1) were silenced with siRNA, mRNA, and protein levels of FAK, mTERF1, and cyclin D1 were determined. HPASMC proliferation increased under hypoxia compared to normal conditions. Knocking down FAK or mTERF1 with siRNA led to decreased cell proliferation under both normal and hypoxia conditions. FAK knockdown led to the reduction of both mTERF1 and cyclin D1 expressions under the hypoxia conditions, whereas mTERF1 knockdown led to the downregulation of cyclin D1 expression but not FAK expression under the same condition. However, under normal conditions, knocking down either FAK or mTERF1 had no impact on cyclin D1 expression. These results suggested that FAK may regulate the mTERF1/cyclin D1 signaling pathway to modulate cell proliferation in hypoxia.

Targeting IL-11 system as a treatment of pulmonary arterial hypertension

IL-11 is linked to fibrotic diseases, but its role in pulmonary hypertension is unclear. We examined IL-11's involvement in idiopathic pulmonary arterial hypertension (iPAH). Using samples from control (n = 20) and iPAH (n = 6) subjects, we assessed IL-11 and IL-11Rα expression and localization through RT-qPCR, ELISA, immunohistochemistry, and immunofluorescence. A monocrotaline-induced PAH model helped evaluate the impact of siRNA-IL-11 on pulmonary artery remodeling and PH. The effects of recombinant human IL-11 and IL-11Rα on human pulmonary artery smooth muscle cell (HPASMC) proliferation, pulmonary artery endothelial cell (HPAEC) mesenchymal transition, monocyte interactions, endothelial tube formation, and precision cut lung slice (PCLS) pulmonary artery remodeling and contraction were evaluated. IL-11 and IL-11Rα were over-expressed in pulmonary arteries (3.2-fold and 75-fold respectively) and serum (1.5-fold and 2-fold respectively) of patients with iPAH. Therapeutic transient transfection with siRNA targeting IL-11 resulted in a significant reduction in pulmonary artery remodeling (by 98%), right heart hypertrophy (by 66%), and pulmonary hypertension (by 58%) in rats exposed to monocrotaline treatment. rhIL-11 and soluble rhIL-11Rα induce HPASMC proliferation and HPAEC to monocyte interactions, mesenchymal transition, and tube formation. Neutralizing monoclonal IL-11 and IL-11Rα antibodies inhibited TGFβ1 and EDN-1 induced HPAEC to mesenchymal transition and HPASMC proliferation. In 3D PCLS, rhIL-11 and soluble rhIL-11Rα do not promote pulmonary artery contraction but sensitize PCLS pulmonary artery contraction induced by EDN-1.In summary, IL-11 and IL-11Rα are more highly expressed in the pulmonary arteries of iPAH patients and contribute to pulmonary artery remodeling and the development of PH.

Inhibition of KIR2.1 decreases pulmonary artery smooth muscle cell proliferation and migration

The investigation of effective therapeutic drugs for pulmonary hypertension (PH) is critical. KIR2.1 plays crucial roles in regulating cell proliferation and migration, and vascular remodeling. However, researchers have not yet clearly determined whether KIR2.1 participates in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and its role in pulmonary vascular remodeling (PVR) also remains elusive. The present study aimed to examine whether KIR2.1 alters PASMC proliferation and migration, and participates in PVR, as well as to explore its mechanisms of action. For the in vivo experiment, a PH model was established by intraperitoneally injecting Sprague-Dawley rats monocrotaline (MCT). Hematoxylin and eosin staining revealed evidence of PVR in the rats with PH. Immunofluorescence staining and western blot analysis revealed increased levels of the KIR2.1, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) proteins in pulmonary blood vessels and lung tissues following exposure to MCT, and the TGF-β1/SMAD2/3 signaling pathway was activated. For the in vitro experiments, the KIR2.1 inhibitor, ML133, or the TGF-β1/SMAD2/3 signaling pathway blocker, SB431542, were used to pre-treat human PASMCs (HPASMCs) for 24 h, and the cells were then treated with platelet-derived growth factor (PDGF)-BB for 24 h. Scratch and Transwell assays revealed that PDGF-BB promoted cell proliferation and migration. Immunofluorescence staining and western blot analysis demonstrated that PDGF-BB upregulated OPN and PCNA expression, and activated the TGF-β1/SMAD2/3 signaling pathway. ML133 reversed the proliferation and migration induced by PDGF-BB, inhibited the expression of OPN and PCNA, inhibited the TGF-β1/SMAD2/3 signaling pathway, and reduced the proliferation and migration of HPASMCs. SB431542 pre-treatment also reduced cell proliferation and migration; however, it did not affect KIR2.1 expression. On the whole, the results of the present study demonstrate that KIR2.1 regulates the TGF-β1/SMAD2/3 signaling pathway and the expression of OPN and PCNA proteins, thereby regulating the proliferation and migration of PASMCs and participating in PVR.

Aldehyde Dehydrogenase 2 (ALDH2) Elicits Protection against Pulmonary Hypertension via Inhibition of ERK1/2-Mediated Autophagy.

Pulmonary hypertension (PH) is caused by chronic hypoxia that induces the migration and proliferation of pulmonary arterial smooth muscle cells (PASMCs), eventually resulting in right heart failure. PH has been related to aberrant autophagy; however, the hidden mechanisms are still unclear. Approximately 40% East Asians, equivalent to 8% of the universal population, carry a mutation in Aldehyde dehydrogenase 2 (ALDH2), which leads to the aggregation of noxious reactive aldehydes and increases the propensity of several diseases. Therefore, we explored the potential aspect of ALDH2 in autophagy associated with PH. In vitro mechanistic studies were conducted in human PASMCs (HPASMCs) after lentiviral ALDH2 knockdown and treatment with platelet-derived growth factor-BB (PDGF-BB). PH was induced in wild-type (WT) and ALDH2-knockout (ALDH2−/−) mice using vascular endothelial growth factor receptor inhibitor SU5416 under hypoxic conditions (HySU). Right ventricular function was assessed using echocardiography and invasive hemodynamic monitoring. Histological and immunohistochemical analyses were performed to evaluate pulmonary vascular remodeling. EdU, transwell, and wound healing assays were used to evaluate HPASMC migration and proliferation, and electron microscopy and immunohistochemical and immunoblot assays were performed to assess autophagy. The findings demonstrated that ALDH2 deficiency exacerbated right ventricular pressure, hypertrophy, fibrosis, and right heart failure resulting from HySU-induced PH. ALDH2−/− mice exhibited increased pulmonary artery muscularization and 4-hydroxynonenal (4-HNE) levels in lung tissues. ALDH2 knockdown increased PDGF-BB-induced PASMC migration and proliferation and 4-HNE accumulation in vitro. Additionally, ALDH2 deficiency increased the number of autophagosomes and autophagic lysosomes together with autophagic flux and ERK1/2-Beclin-1 activity in lung tissues and PASMCs, indicating enhanced autophagy. In conclusion, the study shows that ALDH2 has a protective role against the migration and proliferation of PASMCs and PH, possibly by regulating autophagy through the ERK1/2-Beclin-1 pathway.

Cigarette smoke extract stimulates human pulmonary artery smooth muscle cell proliferation: Role of inflammation and oxidative stress.

Objective(s):Cigarette smoke may play a direct role in proliferation of human pulmonary artery smooth muscle cells (HPASMCs). However, the mechanism involved and the effect of interventions remain unclear. We aimed to evaluate the effect of cigarette smoke extract (CSE) on HPASMCs, explore the role of inflammation and oxidative stress, and the effects of Tempol and PDTC in this process.Materials and Methods:HPASMCs were subjected to normal control (NC), CSE, CSE+Tempol (CSE+T), and CSE+PDTC (CSE+P) groups. Proliferation of HPASMCs was measured by CCK-8 and Western blot. TNF-α, IL-6, MDA, and SOD levels were determined by ELISA and commercial kits. Nuclear translocation of NF-κB p65 was evaluated by western blot.Results:1%, 2.5%, and 5% CSE all promoted proliferation of HPASMCs, and effect of 1% CSE was the most significant, however, 7.5% and 10% CSE inhibited viability of cells (all P<0.05). Compared with the NC group, TNF-α, IL-6, and MDA levels increased, SOD activity decreased (all P<0.05), and NF-κB p65 expression in nuclei increased (P=0.04) in the CSE group. Tempol and PDTC inhibited the proliferation of HPASMCs induced by CSE (all P<0.05). And compared with the CSE group, TNF-α, IL-6, and MDA levels in CSE+T and CSE+P groups decreased, while SOD activity increased (all P<0.05). Tempol reduced the expression of NF-κB p65 in nuclei but did not achieve a significant difference (P=0.08). PDTC inhibited the nuclear translocation of NF-κB p65 (P=0.03).Conclusion:CSE stimulates HPASMCs proliferation in a certain concentration range. The CSE-induced proliferation of HPASMCs involved excessive inflammatory response and oxidative stress. Tempol and PDTC attenuate these effects of CSE on HPASMCs.

Flavopiridol Mitigates the Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Targeting Cyclin-Dependent Kinase 9

PurposeTo investigate the role of cyclin-dependent kinase 9 (CDK9) and the therapeutic potential of a CDK9 inhibitor (flavopiridol) in monocrotaline (MCT)-induced pulmonary hypertension (PH).MethodsFor the in vivo experiments, rats with PH were established by a single intraperitoneal injection of MCT (60 mg/kg). After 2 weeks of MCT injection, rats were then treated with flavopiridol (5 mg/kg, i.p., twice a week) or vehicle for 2 weeks. For the in vitro experiments, human pulmonary artery smooth muscle cells (HPASMCs) were treated with flavopiridol (0.025-1 μM) or vehicle under hypoxic conditions. Hemodynamic recording, right ventricle histology, lung histology, and pulmonary arterial tissue isolation were performed. The expression levels of CDK9, RNA polymerase II, c-Myc, Mcl-1, and survivin were determined by qRT-PCR and western blotting, and the proliferation and apoptosis of rat pulmonary arterial tissues and/or HPASMCs were also assayed.ResultsCompared to the control group, CDK9 was upregulated in pulmonary arterial tissues from MCT-induced PH rats and hypoxic cultured HPASMCs. Upregulation of CDK9 was associated with enhanced phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) at serine-2 (Ser-2), promoting the expression of prosurvival and antiapoptotic proteins (c-Myc, Mcl-1, and survivin). Furthermore, treatment with flavopiridol (5 mg/kg) significantly alleviated pulmonary artery remodeling and partially reversed the progression of MCT-induced PH. Consistently, flavopiridol (0.5 μM) treatment decreased the proliferation and induced the apoptosis of cultured HPASMCs under hypoxic conditions. As a result of CDK9 inhibition and subsequent inhibition of RNA pol II CTD phosphorylation at Ser-2, flavopiridol decreased c-Myc, Mcl-1, and survivin expression in isolated pulmonary small arteries, leading to cell growth inhibition and apoptosis.ConclusionFlavopiridol mitigates the progression of MCT-induced PH in rats by targeting CDK9.

Notch4 mediates vascular remodeling via ERK/JNK/P38 MAPK signaling pathways in hypoxic pulmonary hypertension

BackgroundHypoxic pulmonary hypertension (HPH) is a chronic progressive advanced disorder pathologically characterized by pulmonary vascular remodeling. Notch4 as a cell surface receptor is critical for vascular development. However, little is known about the role and mechanism of Notch4 in the development of hypoxic vascular remodeling.MethodsLung tissue samples were collected to detect the expression of Notch4 from patients with HPH and matched controls. Human pulmonary artery smooth muscle cells (HPASMCs) were cultured in hypoxic and normoxic conditions. Real-time quantitative PCR and western blotting were used to examine the mRNA and protein levels of Notch4. HPASMCs were transfected with small interference RNA (siRNA) against Notch4 or Notch4 overexpression plasmid, respectively. Cell viability, cell proliferation, apoptosis, and migration were assessed using Cell Counting Kit-8, Edu, Annexin-V/PI, and Transwell assay. The interaction between Notch4 and ERK, JNK, P38 MAPK were analyzed by co-immunoprecipitation. Adeno-associated virus 1-mediated siRNA against Notch4 (AAV1-si-Notch4) was injected into the airways of hypoxic rats. Right ventricular systolic pressure (RVSP), right ventricular hypertrophy and pulmonary vascular remodeling were evaluated.ResultsIn this study, we demonstrate that Notch4 is highly expressed in the media of pulmonary vascular and is upregulated in lung tissues from patients with HPH and HPH rats compared with control groups. In vitro, hypoxia induces the high expression of Delta-4 and Notch4 in HPASMCs. The increased expression of Notch4 promotes HPASMCs proliferation and migration and inhibits cells apoptosis via ERK, JNK, P38 signaling pathways. Furthermore, co-immunoprecipitation result elucidates the interaction between Notch4 and ERK/JNK/P38. In vivo, silencing Notch4 partly abolished the increase in RVSP and pulmonary vascular remodeling caused by hypoxia in HPH rats.ConclusionsThese findings reveal an important role of the Notch4-ERK/JNK/P38 MAPK axis in hypoxic pulmonary remodeling and provide a potential therapeutic target for patients with HPH.

Knockdown of HSP110 attenuates hypoxia-induced pulmonary hypertension in mice through suppression of YAP/TAZ-TEAD4 pathway

BackgroundPulmonary hypertension (PH) is a progressive and fatal cardiopulmonary disease characterized by pulmonary vascular remodeling and increased pulmonary vascular resistance and artery pressure. Vascular remodeling is associated with the excessive cell proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). In this paper, the effects of heat shock protein-110 (HSP110) on PH were investigated.MethodsThe C57BL/6 mice and human PASMCs (HPASMCs) were respectively exposed to hypoxia to establish and simulate PH model in vivo and cell experiment in vitro. To HSP110 knockdown, the hypoxia mice and HPASMCs were infected with adeno-associated virus or adenovirus carring the shRNAs (short hairpin RNAs) for HSP110 (shHSP110). For HSP110 and yes-associated protein (YAP) overexpression, HPASMCs were infected with adenovirus vector carring the cDNA of HSP110 or YAP. The effects of HSP110 on PH development in mice and cell proliferation, migration and autophagy of PASMCs under hypoxia were assessed. Moreover, the regulatory mechanisms among HSP110, YAP and TEA domain transcription factor 4 (TEAD4) were investigated.ResultsWe demonstrated that expression of HSP110 was significantly increased in the pulmonary arteries of mice and HPASMCs under hypoxia. Moreover, knockdown of HSP110 alleviated hypoxia-induced right ventricle systolic pressure, vascular wall thickening, right ventricular hypertrophy, autophagy and proliferation of PASMCs in mice. In addition, knockdown of HSP110 inhibited the increases of proliferation, migration and autophagy of HPASMCs that induced by hypoxia in vitro. Mechanistically, HSP110 knockdown inhibited YAP and transcriptional co-activator with PDZ-binding motif (TAZ) activity and TEAD4 nuclear expression under hypoxia. However, overexpression of HSP110 exhibited the opposite results in HPASMCs. Additionally, overexpression of YAP partially restored the effects of shHSP110 on HPASMCs. The interaction of HSP110 and YAP was verified. Moreover, TEAD4 could promote the transcriptional activity of HSP110 by binding to the HSP110 promoter under hypoxia.ConclusionsOur findings suggest that HSP110 might contribute to the development of PH by regulating the proliferation, migration and autophagy of PASMCs through YAP/TAZ-TEAD4 pathway, which may help to understand deeper the pathogenic mechanism in PH development.

Novel Pyrazolo[3,4-b] Pyridine Derivative (HLQ2g) Attenuates Hypoxic Pulmonary Hypertension via Restoring cGKI Expression and BMP Signaling Pathway.

Pulmonary hypertension (PH) is an extremely serious cardiopulmonary disease, finally leading to progressive right ventricular failure and death. Our previous studies have nominated HLQ2g, a pyrazolo[3,4-b] pyridine derivative stimulating soluble guanylate cyclase (sGC), as a new candidate for the treatment of PH, but the specific mechanism is still not clear. The PH model induced by hypoxia was established in rats. Right ventricular systolic pressure (RVSP) was assessed by jugular vein catheterization. RV weight was the index to evaluate RV hypertrophy. The protein levels of cGMP-dependent protein kinase type I (cGKI), bone morphogenetic protein receptor 2 (BMPR2), phosphorylated Smad1/5/8 (p-Smad1/5/8), and inhibitor of differention 1 (Id1) in pulmonary artery and human pulmonary artery smooth muscle cells (HPASMCs) were determined by western blotting. Cell proliferation and migration were evaluated. In the whole experiment, the first clinically available sGC stimulator Riociguat was used as the reference. In hypoxic PH rat model, elevated RVSP and RV hypertrophy were significantly reduced by HLQ2g treatment. Both Riociguat and HLQ2g attenuated vascular remodeling accompanied with up-regulated cGKI expression and BMP signaling pathway, which was characterized by elevated expression of BMPR2, p-Smad1/5/8, and Id1 in HPH rats. In addition, HLQ2g inhibited proliferation and migration of HPASMCs induced by hypoxia and platelet-derived growth factor (PDGF), restored BMPR2 signaling, which was recalled by Rp-8-Br-PET-cGMPS, the inhibitor of cGKI. In summary, the novel pyrazolo[3,4-b] pyridine derivative HLQ2g can alleviate HPH progression by up-regulating cGKI protein and BMP signaling pathway.

Galectin-3 participates in PASMC migration and proliferation by interacting with TGF-β1

Pulmonary vascular remodelling is one of the most important factors for pulmonary hypertension (PH). Galectin-3 (Gal-3) is a β-galactoside-binding lectin. In the latest literature, Gal-3 has been reported to be involved in pulmonary vascular remodelling, and its underlying mechanism is unclear. Our research aims to prove the effect of Gal-3 on the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMC) induced by transforming growth factor β1 (TGF-β1) and to study its mechanism. In vivo experiment: In Sprague-Dawley (SD) rats, monocrotaline was injected intraperitoneally to establish a PH model, and the Gal-3 inhibitor (modified citrus pectin, MCP) 28 Ds was administered in the stomach. The results indicate that Gal-3 and TGF-β1 may be involved in the occurrence and development of PH, which may be related to the Smad2/3 signalling pathway. In vitro experiment: Human pulmonary artery smooth muscle cells were pretreated with the Gal-3 inhibitor (MCP) for 24 h, then TGF-β1 or Gal-3 was administered to the cells for 24 h. The results show that exogenous TGF-β1 and Gal-3 can activate the downstream Smad2/3 signalling pathway, and increase the proliferation and migration ability of HPASMC. However, the Gal-3 inhibitor (MCP) inhibited these effects. Further results display that TGF-β1 and Gal-3 could mutually regulate the protein and mRNA expression levels. In summary, the results of this study indicate that Gal-3 regulates the Smad2/3 signalling pathway through protein interaction with TGF-β1, in turn regulates the proliferation and migration of HPASMC, thereby regulating the occurrence and development of PH.

AAV1-Mediated shRNA Knockdown of SASH1 in Rat Bronchus Attenuates Hypoxia-Induced Pulmonary Artery Remodeling.

Pulmonary hypertension (PH) is a proliferative disease characterized by pulmonary arterial remodeling (PAR). SAM and SH3 domain containing 1 (SASH1) is a novel tumor suppressor gene whose biological function in PH is unclear. In this study, a hypoxia-induced pulmonary hypertension (HPH) rat model was constructed to explore the role of SASH1 in PAR. Histopathological changes in the lung tissue and hemodynamic alteration were detected in SASH1-knockdown rats through adeno-associated virus type-1 (AAV1) infection. In vitro, primary human pulmonary arterial smooth muscle cells (HPASMCs) were transfected with SASH1siRNA to investigate the effects of SASH1 on hypoxia-induced proliferation and migration. The molecular mechanisms associated with SASH1 were explored through knockdown and overexpression approaches. We found that SASH1 expression was significantly increased in rat pulmonary arteries and HPASMCs after hypoxia exposure. In vivo, silencing the SASH1 gene expression improved HPH in rats. The SASH1 downregulation inhibited proliferation and migration of hypoxia-induced HPASMCs. The protein expression of phospho-AKT (known as protein kinase B), proliferating cell nuclear antigen, and matrix metalloproteinase 9 (MMP9) in HPASMCs were increased after SASH1 overexpression, whereas these effects were inhibited by SASH1 knockdown. In conclusion, SASH1 downregulation improved hypoxia-induced PAR and PH. SASH1 may be a novel target for PH gene therapy in the era of precision medicine.

CYLD mediates human pulmonary artery smooth muscle cell dysfunction in congenital heart disease-associated pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a common complication of congenital heart disease (CHD). Deubiquitinase cylindromatosis (CYLD) has been reported to significantly aggravate vascular smooth muscle cell (VSMC) phenotypic transformation, proliferation, and migration. Here, we aimed to further investigate its roles and underlying mechanisms in the CHD-PAH development. The expression of CYLD in the lung tissues from CHD-PAH patients and monocrotaline (MCT) plus aortocaval (AV)-induced PAH rats, pulmonary artery smooth muscle cells (PASMCs) from MCT-AV-induced PAH rats, and human PASMCs (HPASMCs) was evaluated. After infection with CYLD siRNA or pcNDA3.1-CYLD, the proliferation, migration, and apoptosis of HPASMCs were measured using an EdU assay, transwell and scratch wound healing assays, and flow cytometric assay, respectively. An adeno-associated virus (AAV) vector encoding CYLD was used to suppress CYLD expression by being intratracheally instilled in rats 7 days before MCT-AV treatment. The results showed that CYLD was increased in the lung tissues from CHD-PAH patients and MCT-AV-induced PAH rats, and in PASMCs from MCT-AV-induced PAH rats. The contractile-type HPASMCs expressed low levels of CYLD, while the proliferative synthetic-type HPASMCs expressed high levels of CYLD. In addition, CYLD could mediate HPASMC dysfunction, which regulated HPASMC phenotypic transformation and proliferation via the modulation of p38 and ERK activation, while CYLD regulated HPASMC migration via the modulation of p38 activation. In vivo results demonstrated that the local suppression of CYLD expression could attenuate the increased levels of PAH and its associated pulmonary vascular remodeling in MCT-AV-induced PAH rats. Collectively, these results indicated that CYLD might be a potential novel therapeutic target for the prevention of PAH and pulmonary vascular remodeling in CHD-PAH through the modulation of HPASMC dysfunction.

Astragaloside IV blocks monocrotaline‑inducedpulmonary arterial hypertension by improving inflammation and pulmonary artery remodeling.

Pulmonary arterial hypertension (PAH) is associated with increased inflammation and abnormal vascular remodeling. Astragaloside IV (ASIV), a purified small molecular saponin contained in the well-know herb, Astragalus membranaceus, is known to exert anti-inflammatory and anti-proliferation effects. Thus, the present study investigated the possible therapeutic effects of ASIV on monocrotaline (MCT)-induced PAH. Rats were administered a single intraperitoneal injection of MCT (60 mg/kg), followed by treatment with ASIV at doses of 10 and 30 mg/kg once daily for 21 days. Subsequently, right ventricle systolic pressure, right ventricular hypertrophy and serum inflammatory cytokines, as well as pathological changes of the pulmonary arteries, were examined. The effects of ASIV on the hypoxia-induced proliferation and apoptotic resistance of human pulmonary artery smooth muscle cells (HPASMCs) and the dysfunction of human pulmonary artery endothelial cells (HPAECs) were evaluated. MCT elevated pulmonary artery pressure and promoted pulmonary artery structural remodeling and right ventricular hypertrophy in the rats, which were all attenuated by both doses of ASIV used. Additionally, ASIV prevented the increase in the TNF-α and IL-1β concentrations in serum, as well as their gene expression in lung tissues induced by MCT. In in vitro experiments, ASIV attenuated the hypoxia-induced proliferation and apoptotic resistance of HPASMCs. In addition, ASIV upregulated the protein expression of p27, p21, Bax, caspase-9 and caspase-3, whereas it downregulated HIF-1α, phospho-ERK and Bcl-2 protein expression in HPASMCs. Furthermore, in HPAECs, ASIV normalized the increased release of inflammatory cytokines and the increased protein levels of HIF-1α and VEGF induced by hypoxia. On the whole, these results indicate that ASIV attenuates MCT-induced PAH by improving inflammation, pulmonary artery endothelial cell dysfunction, pulmonary artery smooth muscle cell proliferation and resistance to apoptosis.

Involvement of TRPC1 and Cyclin D1 in Human Pulmonary Artery Smooth Muscle Cells Proliferation Induced by Cigarette Smoke Extract.

Cigarette smoking contributes to the development of pulmonary artery hypertension (PAH). As the basic pathological change of PAH, pulmonary vascular remodeling is considered to be related to the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs). However, the molecular mechanism underlying this process remains not exactly clear. The aim of this research was to study the molecular mechanism of PASMCs proliferation induced by smoking. Human PASMCs (HPASMCs) were divided into 6 groups: 0% (control group), cigarette smoking extract (CSE)-treated groups at concentrations of 0.5%, 1%, 2%, 5%, 10% CSE respectively. HPASMCs proliferation was observed after 24 h. HPASMCs were divided into two groups: 0 (control group), 0.5% CSE group. The mRNA and protein expression levels of transient receptor potential channel 1 (TRPC1) and cyclin D1 in HPASMCs after CSE treatment were respectively detected by RT-PCR and Western blotting. The intracellular calcium ion concentration was measured by the calcium probe in each group. In the negative control group and TRPC1-siRNA transfection group, the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein were detected. Data were compared with one-way ANOVA (for multiple-group comparison) and independent t-test (for two-group comparison) followed by the least significant difference (LSD) test with the computer software SPSS 17.0. It was found that 0.5% and 1% CSE could promote the proliferation of HPASMCs (P<0.05), and the former was more effective than the latter (P<0.05), while 3% and above CSE had inhibitory effect on HPASMCs (P<0.05). The mRNA and protein expression levels of TRPC1 and cyclin D1 in 0.5% and 1% CSE groups were significantly higher than those in the control group (P<0.05), while those in 3% CSE group were significantly decreased (P<0.05). Moreover, the proliferation of HPASMCs and the expression of cyclin D1 mRNA and protein in TRPC1-siRNA transfection group were significantly reduced as compared with those in the negative control group (P<0.05). It was concluded that low concentration of CSE can promote the proliferation of HPASMCs, while high concentrations of CSE inhibit HPASMCs proliferation. These findings suggested that CSE induced proliferation of HPASMCs at least in part via TRPC1-mediated cyclin D1 expression.

Nestin represents a potential marker of pulmonary vascular remodeling in pulmonary arterial hypertension associated with congenital heart disease

ObjectiveReportedly, nestin was re-expressed in proliferative synthetic-type pulmonary artery smooth muscle cells (PASMCs) and obligatory for PASMC proliferation in pulmonary arterial hypertension (PAH). Accordingly, nestin is increased in pulmonary vascular lesions of congenital heart disease (CHD)-associated PAH patients. We tested the hypothesis whether nestin was re-expressed in proliferative synthetic-type PASMCs and associated with pulmonary vascular remodeling in CHD-PAH. Materials and methodsNestin expression was tested using lung tissues from CHD-PAH patients and monocrotaline (MCT) plus aortocaval (AV) shunt-induced PAH rats, human PASMCs (HPASMCs), and pulmonary artery endothelial cells (PAECs) and PASMCs from MCT-AV-induced PAH rats. The role and possible mechanism of nestin on HPASMC proliferation, apoptosis, cell cycle and migration were investigated by assays of CCK-8, EdU, TUNEL, flow cytometry, transwell chamber and immunoblotting assays. ResultsNestin was solely expressed in proliferative synthetic-type PASMCs, but rarely detected in PAECs. Nestin was barely detected in normal pulmonary arterioles and occlusive pulmonary vascular lesions. Its expression was robustly increased in developing pulmonary vasculature, but returned to normal levels at the late stage of pulmonary vascular remodeling in lung tissues from CHD-PAH patients and MCT-AV-induced PAH rats. Besides, nestin peaks were consistent with the histological features in lung tissues of MCT-AV-induced PAH rats. Moreover, nestin overexpression effectively promoted HPASMC phenotypic transformation, proliferation, apoptosis resistance and migration via enhancing Wnt/β-catenin activation. ConclusionsThese data indicated that nestin was re-expressed in proliferative synthetic-type PASMCs and might represent a potential marker of pulmonary vascular remodeling in CHD-PAH.

Identification of Key Players Involved in CoCl2 Hypoxia Induced Pulmonary Artery Hypertension in vitro.

BackgroundThe proliferation of human pulmonary artery smooth muscle cells (HPASMCs) induced by hypoxia was considered as the main cause of pulmonary arterial hypertension (PAH). This study aimed to explore potential genes and long non-coding RNAs (lncRNAs) involved in the mechanism of hypoxia-induced PAH.MethodsCoCl2 was utilized to induce hypoxia in HPASMCs, and then cell proliferation, apoptosis, and expression of hypoxia-inducible factors (HIF)-1α were determined. Meanwhile, the RNA isolated from CoCl2-treated cells and control cells were sequenced and differentially expressed genes/lncRNA (DEGs/DELs) were screened, followed by protein-protein interaction (PPI) construction, functional enrichment analyses, and lncRNA-target prediction. Finally, the expression of key genes and lncRNAs were validated using quantitative real-time PCR and western blotting.ResultsCoCl2 treatment could significantly increase the expression of HIF-1α and the proliferation of HPASMCs. A total of 360 DEGs and 57 DELs were identified between CoCl2 treated and control cells. Functional enrichment analysis showed that up-regulated DEGs and DELs’ targets, including LDHA, PFKP, and VEGFA, were significantly enriched in biological processes related to hypoxia or oxygen levels, and the downregulated DEGs and DELs’ targets were significantly enriched in extracellular-matrix-related biological processes. In addition, LDHA, PFKP, and VEGFA exhibited a strong relationship with miR-100HG and TSPEAR-AS2 in lncRNA-target network. The protein level of LDHA, PFKP, and VEGFA were all increased.ConclusionLDHA, PFKP, VEGFA, and lncRNA miR-100HG and TSPEAR-AS2 probably played crucial roles in the pathogenesis of CoCl2 hypoxia-induced-HAP, which might serve as promising therapeutic targets for PAH.

Berberine alleviates pulmonary hypertension through Trx1 and β-catenin signaling pathways in pulmonary artery smooth muscle cells

Pulmonary arterial hypertension (PAH) is closely associated with profound vascular remodeling, especially pulmonary arterial medial hypertrophy and muscularization, due to aberrant proliferation of pulmonary artery smooth muscle cells (PASMCs). Berberine, a drug commonly used to treat inflammation, may be a novel therapeutic option for PAH by improving pulmonary artery remodeling. The present study investigated whether berberine affected Trx1/β-catenin expression and/or activity and whether it could reduce the development of pulmonary hypertension in an experimental rat model and proliferation in human PASMCs (HPASMCs). The results showed that increased proliferation in hypoxia-induced healthy PASMCs or PAH PASMCs was associated with a significant increase in Trx1 and β-catenin expression. Treatment with the Trx1-specific inhibitor PX-12 significantly reduced pulmonary arterial pressure and vascular remodeling, as well as improved in vivo cardiac function and right ventricular hypertrophy, in Su/Hox-induced PAH rats. Berberine reversed right ventricular systolic pressure and right ventricular hypertrophy and decreased pulmonary vascular remodeling in the rats. Furthermore, berberine had an antiproliferative effect on hypoxia-induced HPASMC proliferation in a manner likely mediated by inhibiting Trx1 and its target gene β-catenin expression. Our work will help elucidate novel strategies for PAH treatment involving the traditional Chinese medicine berberine, and Trx1/β-catenin may be a promising therapeutic target.

Inhibition of RELM-β prevents hypoxia-induced overproliferation of human pulmonary artery smooth muscle cells by reversing PLC-mediated KCNK3 decline

AimsAlthough resistin-like molecule β (RELM-β) is involved in the pathological processes of various lung diseases, such as pulmonary inflammation, asthma and fibrosis, its potential roles in hypoxic pulmonary arterial hypertension (PAH) remain largely unknown. The study aims to investigate whether RELM-β contributes to hypoxia-induced excessive proliferation of human pulmonary artery smooth muscle cells (PASMCs) and to explore the potential mechanisms of this process. Main methodsHuman PASMCs were exposed to normoxia or hypoxia (1% O2) for 24 h. siRNA targeting RELM-β was transfected into cells. Protein levels of KCNK3, RELM-β, pSTAT3 and STAT3 were determined by immunoblotting. The translocation of NFATc2 and expression of KCNK3 were visualized by immunofluorescence. 5-ethynyl-2′-deoxyuridine assays and cell counting kit-8 assays were performed to assess the proliferation of PASMCs. Key findings(1) Chronic hypoxia significantly decreased KCNK3 protein levels while upregulating RELM-β protein levels in human PASMCs, which was accompanied by excessive proliferation of cells. (2) RELM-β could promote human PASMCs proliferation and activate the STAT3/NFAT axis by downregulating KCNK3 protein under normoxia. (3) Inhibition of RELM-β expression effectively prevented KCNK3-mediated cell proliferation under hypoxia. (4) Phospholipase C (PLC) inhibitor U-73122 could not only prevent the hypoxia/RELM-β-induced decrease in KCNK3 protein, but also inhibit the enhanced cell viability caused by hypoxia/RELM-β. (5) Both hypoxia and RELM-β could downregulate membrane KCNK3 protein levels by enhancing endocytosis. SignificanceRELM-β activation is responsible for hypoxia-induced excessive proliferation of human PASMCs. Interfering with RELM-β may alleviate the progression of hypoxic PAH by upregulating PLC-dependent KCNK3 expression.

Extracellular Superoxide Dismutase Regulates Early Vascular Hyaluronan Remodeling in Hypoxic Pulmonary Hypertension

Chronic hypoxia leads to pathologic remodeling of the pulmonary vasculature and pulmonary hypertension (PH). The antioxidant enzyme extracellular superoxide dismutase (SOD3) protects against hypoxia-induced PH. Hyaluronan (HA), a ubiquitous glycosaminoglycan of the lung extracellular matrix, is rapidly recycled at sites of vessel injury and repair. We investigated the hypothesis that SOD3 preserves HA homeostasis by inhibiting oxidative and enzymatic hyaluronidase-mediated HA breakdown. In SOD3-deficient mice, hypoxia increased lung hyaluronidase expression and activity, hyaluronan fragmentation, and effacement of HA from the vessel wall of small pulmonary arteries. Hyaluronan fragmentation corresponded to hypoxic induction of the cell surface hyaluronidase-2 (Hyal2), which was localized in the vascular media. Human pulmonary artery smooth muscle cells (HPASMCs) demonstrated hypoxic induction of Hyal2 and SOD-suppressible hyaluronidase activity, congruent to our observations in vivo. Fragmentation of homeostatic high molecular weight HA promoted HPASMC proliferation in vitro, whereas pharmacologic inhibition of hyaluronidase activity prevented hypoxia- and oxidant-induced proliferation. Hypoxia initiates SOD3-dependent alterations in the structure and regulation of hyaluronan in the pulmonary vascular extracellular matrix. These changes occurred soon after hypoxia exposure, prior to appearance of PH, and may contribute to the early pathogenesis of this disease.